Willheim M, Agis H, Sperr W R, Köller M, Bankl H C, Kiener H, Fritsch G, Füreder W, Spittler A, Graninger W, Scheiner O, Gadner H, Lechner K, Boltz-Nitulescu G, Valent P
Institute of General and Experimental Pathology, University of Vienna, Neubau AKH, Austria.
J Immunol Methods. 1995 May 11;182(1):115-29. doi: 10.1016/0022-1759(95)00034-8.
Basophils and mast cells represent distinct cell lineages within the hemopoietic system. Based on the unique cell surface antigen profile of both cells, we have established methods which allow the reproducible purification to homogeneity (> 99%) of normal human basophil granulocytes from the peripheral blood and of mast cells from human dispersed tissues. Basophils (n = 9) were purified by current counterflow elutriation followed by depletion of monocytes with CD14 mAb conjugated to magnetic beads, and subsequent cell sorting for CD217+ cells. Basophil purity was 99.5 +/- 0.4% (range 98.7-99.9%). Mast cells were obtained from lung (n = 6), uterus (n = 1), mastocytosis bone marrow (n = 2), and human foreskin (n = 2). Mast cells were purified by collagenase digestion followed by current counterflow elutriation and sorting with CD117/c-kit mAb. Mast cell purity was 99.4 +/- 0.7% (range: 97.5-99.9%). Purified cells were more than 90% viable and were able to release histamine on induction with IgE plus anti-IgE. Furthermore, the PCR technique could be applied on pure cells and confirmed expression of high affinity IgE receptor (Fc epsilon R1) alpha chain mRNA. Thus, by combining isolation techniques including elutriation, magnetic cell depletion and cell sorting with mAb, functionally intact normal human basophils and mast cells can be enriched to homogeneity.
嗜碱性粒细胞和肥大细胞是造血系统中不同的细胞谱系。基于这两种细胞独特的细胞表面抗原谱，我们建立了一些方法，能够将正常人外周血嗜碱性粒细胞和人分散组织中的肥大细胞可重复地纯化至均一性（>99%）。通过连续逆流淘析法纯化嗜碱性粒细胞（n = 9），随后用与磁珠偶联的CD14单克隆抗体去除单核细胞，然后对CD217+细胞进行细胞分选。嗜碱性粒细胞纯度为99.5±0.4%（范围98.7 - 99.9%）。肥大细胞取自肺（n = 6）、子宫（n = 1）、肥大细胞增多症骨髓（n = 2）和人包皮（n = 2）。通过胶原酶消化、连续逆流淘析法以及用CD117/c-kit单克隆抗体进行分选来纯化肥大细胞。肥大细胞纯度为99.4±0.7%（范围：97.5 - 99.9%）。纯化后的细胞活力超过90%，并且在IgE加抗IgE诱导下能够释放组胺。此外，PCR技术可应用于纯细胞，并证实了高亲和力IgE受体（FcεR1）α链mRNA的表达。因此，通过将包括淘析、磁性细胞去除和单克隆抗体细胞分选在内的分离技术相结合，可以将功能完整的正常人嗜碱性粒细胞和肥大细胞富集至均一性。
